In phase I we have identified a new class of active-site directed inhibitors of DMA polymerase HIE, a novel replicative enzyme in Gram+ bacteria. The compounds, 7-substituted-N2-(3,4-dichlorobenzyl)guanines "DCBGs", are potent enzyme inhibitors, and selected derivatives have potent and broad activity against clinically relevant Gram+ bacteria. We have identified the first active site directed inhibitors of the related DNA polymerase HIE from Gram- bacteria, i.e. E. coli. In addition, we have established a collaboration to crystallize and solve the structure of a complex between E. fecalis pol IIIE, DNA and one of our inhibitors. [unreadable] Based on the results of phase I, we will pursue designation of a lead antibiotic compound by in vivo testing in systemic and toplical bacterial infection models. We will use both QSAR analysis and structure-based drug design to discover new platform inhibitors of the pol HIE target. The following specific aims will be pursued: [unreadable] 1. scale-up synthesis of lead compound candidates and formulations for evaluation in animal models of bacterial infection; synthesis of 7-substituted analogs with enhanced pol HIE inhibitory and antibacterial activity in vitro. [unreadable] 2. solve the structure of E. fecalis pol IIIE:DNA:inhibitor complexes, and use the coordinates for understanding the basis of inhibition of the enzyme and for further rational, computer-based drug design (collaboration with Dr. Mark Jedrzejas, Children's Hospital of Oakland Research Institute). [unreadable] 3. continue assays of compounds for enzyme inhibition (pol IIIC, pol HIE), antibacterial activity, selectivity, cytotoxicity; incidence of resistance and mechanism(s) of resistance, by cloning and sequencing of targets (by subcontract to Microbiotix Inc.); test candidate drugs against clinical isolates of Gram+ and Gram- bacteria, bactericidal assays, combination studies with marketed antibiotics (by subcontract to UMass Medical School). [unreadable] 4. develop analytical methods for analysis of candidates in animal plasma, drug uptake and distribution by various routes in mice, acute toxicity, in vitro metabolism and stability studies. [unreadable] 5. evaluate candidate drugs for activity in animal models of Gram+ and Gram- infections (if warranted), emphasizing systemic antibiotic-resistant S. aureus, E. fecalis and S. pneumoniae in mice and topical S. aureus infections in guinea pigs.. [unreadable] Potent inhibition of Gram+ pol HIE and pol IIIC may lead to antibiotics with reduced incidence of resistance. [unreadable] [unreadable]